Viral-transduced gene expression is the current standard for cell-type-specific labeling and cell tacking in experimental neuroscience. To achieve widespread gene expression, a viral delivery method to neonatal rodents was introduced more than two decades ago. Most of those neonatal viral vector injection-based gene transduction methods in mice used deep hypothermia for anesthesia, which was reported to be associated with behavioral impairments. To explore other options for neonatal viral applications, we applied a combination of Medetomidine, Midazolam, and Fentanyl (MMF), each of which can be antagonized by a specific antagonist. Later in their adulthood, we found that adult mice, that received the MMF-induced anesthesia, combined with virus-injected into the brain at postnatal day 2, showed similar performance in all behavioral tasks tested, including tasks for motor coordination, anxiety-related tasks, and spatial memory when compared to adult naïve littermates. This demonstrates that MMF anesthesia could be safely applied to mice for neonatal viral transduction at P2. Classical virus-mediated gene transfer approaches into pre-selected brain regions are mostly limited to adult animals n Saito, , or systn Saito,  achievedTherefore, we used the neonatal rAAV gene delivery method in a pilot study with cryoanesthesia, also referred to as hypothermia, to establish a mouse model for autism spectrum disorder  bind to GABAA receptors, while Fentanyl and its antagonist Naloxone (N-Narcanti) act on μ-opioid receptors. These drugs and their antagonists can be injected either intramuscularly or subcutaneously. The half-life of these compounds is between 0.5 and 3.0 h. Although this combination is one of the well-established anesthetic techniques, it is not yet commonly used and not everywhere accepted for small rodents. Moreover, it is not known how this pharmacological intervention that takes place shortly after birth affects later the behavior and cognitive performance of those mice.We received the governmental permission to investigate the application and long-term effects of a “new” set of general anesthesia drugs in newborn mice. We decided to use a the combination of Medetomidine (M-Domitor), Midazolam (M-Dormicum) and Fentanyl , since (I) each of the three drugs can be antagonized by a very specific antagonist, and (II) MMF anesthesia had been used for rabbits and other mammals  to the neonatal C57BL/6N and FVB/N mice and determined the earliest time point for successful pharmacological MMF-AFN anesthesia combined with rAAV injection. Later in adulthood of MMF-AFN/rAAV anesthetized mice, the locomotion, anxiety and cognitive performance were evaluated.All experimental procedures were performed according to the animal welfare guidelines of the Max Planck Society and were in accordance with the German Animal Protection Law (TierSCHG). The Animal Ethics Committee of the MPImF/Heidelberg approved all experimental procedures and the regional veterinary authorities in Karlsruhe, Germany (License Number: 35-9185.82/A-38/10) accepted and supported the experimental protocol. The project was in accordance with the European 2006 Convention ETS 123 EG and Parliament/Council Directive 2010/63/EU. Legal access to drug was approved by the BfAM license to UZ.SYN-Venus. Up to 30 min after MMF treatment, the AFN antagonists [50 μl Atipamezol  + 500 μl Flumazenil  + 300 μl Naloxon ] were injected subcutaneously into the right hip (8.5 μl/g body weight). The pups recovered about 3 min after the application of the AFN mixture.Pregnant embryonic day 16 (E16) C57BL/6N and FBV/N mice were purchased from Charles River. Pups were born 5–6 days later at the animal facility of the Max Planck Institute for Medical Research. On the day of treatment, the postnatal day 0–2 (P0–P2) pups were first removed from the home cages and their dams. Pups were immediately anesthetized with a mixture of Medetomidine (M-Domitor), Midazolam (M-Dormicum) and Fentanyl (F-Fentanyl) [MMF mixture], which was prepared by 50 μl Medetomidine , 100 μl Midazolam  and 100 μl Fentanyl . Except controls, each pup was injected subcutaneously in the area of the left hip with 2.5 μl MMF/g body weight . For the P2 pups, 5–10 min after MMF application, the deeply anesthetized animals were injected with serotype 1/2 rAAV-SYN-Venus virus stocks for neuronal labeling by the rAAV human synapsin promoter . For neonatal injection .Serotype 1/2 rAAV-After injection and recovery from anesthesia, pups were transferred to their home cages with their dams. Members of our research team monitored the physical condition of all animals 2–3 times per day until P8. At P28, offspring were separated from their mothers and housed with 1–2 sex-matched littermates/cage. At the age of 4 months, animals were transferred to individually ventilated cages in the animal room. The type II cages were installed in two-pipe system racks (Biozone). The cages were equipped with clean, fresh water and dust reduced wood bedding (Rettenmeier). The racks were individually ventilated by their own integrated ventilation systems (approx. negative pressure 5 Pa at 100 air exchanges/h). Room temperature in all animal rooms was kept at 20–24°C/relative humidity of 40–60% and constant lighting from 6:00 a.m. to 6:00 p.m. at 150–200 lux.2HPO4/ 2H2O, 1.4 mM KH2PO4; all from Sigma-Aldrich) and 4% paraformaldehyde  in PBS prior to decapitation. Brains were removed and after post-fixation in ice-cold 4% PFA for 2 h each brain was embedded in 2.5% agarose (Invitrogen)/PBS. Agarose embedded brains were sliced (70 μm sections) on a vibratome . To increase the contrast of the Venus signal in the thick vibratome sections, the Venus fluorescence was visualized together with its immunoreactivity by using a rabbit anti-GFP antibody  and FITC-coupled anti-rabbit secondary antibody . For the NeuN staining, mouse anti-NeuN antibody were used  and followed with Cy3-coupled anti-mouse secondary antibody . Immunstaining was performed as described  and perfused intracardially with PBS ; (ii) MMF-AFN anesthesia (MMF-AFN–V n = 10); and (iii) naïve controls (n = 7). In total four litters of C75BL6/N mice were selected for the behavioral tests. Before the behavioral test started, 2 MMF-AFN treated animals died, and 1 MMF-AFN + rAAV-SYN-Venus treated animal was excluded from the pre-handling due to hyperactivity . The order of those 23 single-housed animals was randomized for the experimenter. The experimenter was blind to the history of each of those 23 C57BL6/N mice.Behavioral experiments were performed with 3-month-old animals during the light cycle. Each behavioral test and pre-handling day, the home-caged mice were moved first to the animal test room for 1 h of habituation before the behavioral challenge started. Pre-handling with each mouse was performed for 3 days by the experimenter as described Deacon, . The ordFor the quantification of nesting behavior, mice were single-housed overnight in cages with 20 × 20 cm square sized white tissues as nesting material. At the next morning, nest construction was scored according to the following scheme: 1 = no nesting; 2 = some craters in bedding; 3 = nestlets mostly shredded but not identifiable nest site; 4 = an identifiable but flat nest; 5 = nestlets pulled to pieces and closed roof formed, covering the mouse completely. Higher scores in this task is associating with intact hippocampal processing Deacon, .Mice were suspended by their tails for 6 min. The body and limb posture, as well as immobility and amount of movements, were assessed in 30 s intervals  had five 3 cm diameter drums, suitably machined to provide grip. The mouse was held by the tail and released as soon as the mouse could grip the rotating rod, which was rotating at a speed of 4 rpm. The rod rotation was gradually accelerated until it reached the maximum of 40 rpm. If the mouse fell off the rod, the time was recorded as latency to fall of the Rota-Rod . The time, it took each animal to reach the other end of the 50 cm long bar was recorded Deacon, .The self-constructed Elevated Plus-Maze was elevated 100 cm from the floor. The Plus-Maze was composed of two opposing 50 × 7 cm wide closed arms  and two opposing 50 cm long open arms  and a neighboring 15 × 20 × 20 cm closed dark compartment. Both compartments were connected by an open door 3 × 3 cm. The mouse was placed in the middle of the light compartment facing away from the opening to the dark compartment. The number of transitions between the light and dark compartment as well as the total time spent on the dark compartment of an animal was measured in one 5 min test run . Mice were placed in a corner of an open field and allowed to explore the novel environment for 10 min. The movement and the position of the animal was video-taped from the top. After 10 min, a novel object (LEGO TOY) was placed in the center of the open field. The time, which the mice spent in the center, was monitored during first 10 min (without object) and the next 5 min when the novel object was presented.The T-Maze test is a spatial working memory (SWM) test, analyzing the animals’ ability to alternate between a new unknown and a familiar environment based on their memory of the previously visited arms , in which the animals learn to choose the rewarded goal arm based on the position of the extra maze cues . One-way ANOVA was performed for the body weight, Elevated Plus-Maze test, the Dark-Light-Box test and the open field activity test; two-way repeated measurement ANOVA was performed for Rota-Rad test, Horizontal Bar test, T-Maze and Y-Maze tests. n = 14) were successfully anesthetized up to 30 min and recovered 3–5 min after antagonist injection. After the recovery from anesthesia, pups were placed back to their home cages. However, 12–24 h after MMF-ANF administration, all MMF-AFN treated neonatal mice had died. Similarly, the pups treated with MMF-AFN during the 24–48 h time window (P1) after birth (n = 15) did not survive the MMF-AFN treatment. Only when the MMF-AFN anesthesia was administrated during 48–72 h postnatal window (P2) all anesthetized pups (n = 10) recovered from the temporal MMF-AFN anesthesia. It is known that the MMF-AFN administration might affect the animals’ cardiorespiratory system  of the main olfactory bulb (SYN-Venus-transduced subventricular zone (SVZ) stem cells and that the human synapsin promoter fragment of the rAAV-SYN-Venus is already active in those neuronal progenitor cells. NeuN and anti-GFP positive neurons are located outside the center of the olfactory bulb. Most likely, they represent terminally-differentiated mature granule cells derived from rAAV-transduced migrating neuronal stem cells of the SVZ , MMF-AFN–Virus (n = 8) and MMF-AFN+Virus (n = 8) animals. Eleven weeks post-delivery, mice of those three groups were single-housed for 1 week before the pre-handling and the behavioral tests started. When analyzed for the home-cage behavior, mice of all three groups exhibited regular nesting score and tail suspension (data not shown), and had comparable body weight at the age of 12 weeks  = 0.5006, p = 0.6136). Motor coordination of the mice was analyzed in the accelerating Rota-Rod and the Horizontal Bar tests. All animals of the three cohorts performed equally well in the Rota-Rod, and Two-way repeated measurement ANOVA showed no significant difference among all groups  = 0.6623, p = 0.5266). Similar results were found in the Horizontal Bar test  = 1.228, p = 0.3141). Thus, neither the home-cage behavior nor the simple or complex motor coordination tests provided any indication for potential long-term general neuronal dysfunction induced by MMF-AFN treatment and/or the combined rAAV injection at P2.To analyze whether the MMF-AFN anesthesia in P2 pups will lead to behavioral impairments in the adults, all P2 test animals were separated from their mothers and caged in sex-matched groups at P28. Offspring from C57BL/6N dams were randomly categorized into three groups: untreated, naïve control animals  = 0.9771, p = 0.3937). Similarly, in the Dark-Light-Box, the total time spent in the dark compartment was comparable  = 1.384, p = 0.2736) and the number of transitions between the light and dark compartment showed no significant difference among groups  = 1.945, p = 0.1691). To further investigate the response to novel environments and objects, locomotion and object exploration were monitored in the open field (post hoc test (Bonferroni’s Multiple Comparison Test) showed no significant difference among three groups  = 0.5163, p = 0.3064), demonstrating normal locomotion of the P2 MMF-AFN-treated animals in comparison to naïve mice. After introducing a novel object (NO) in the center of the open field, all mice spent more time in the center to explore this object compared the time that they spent in the outer quadrants of the open field . Thus, the NO exploration ability was also not affected by the MMF-AFN treatment, combined with injections at P2.The anxiety-related behavior was monitored in the Elevated Plus-Maze and the Dark-Light-Box. The animals from MMF-AFN anesthetized with rAAV-injected group and only MMF-AFN anesthetized group showed no increased anxiety-related behavior when compared to untreated mice in Elevated Plus-Maze. The time the mice spent in the closed arms was not significantly different among mice of the three groups  = 1.074, p = 0.3886, treatment F = 1.229, p = 0.3148, and trials F = 1.525, p = 0.2179). Post hoc tests (Bonferroni test) demonstrated also no difference on each experimental day between either two of the three groups (data not shown). Thus, SWM was not changed by rAAV injections under MMF-AFN anesthesia at P2.To analyze higher cognitive function in P2-MMF-AFN anesthetized and rAAV-injected mice, we first examined the SWM in the T-Maze in the 3 months old P2-MMF-AFN anesthetized and rAAV-injected mice  = 15.03, p < 0.0001). And two-way repeated measurement ANOVA showed no difference among all groups  = 2.049, p = 0.1563). Post hoc tests (Boferroni test) indicated no difference on each experimental day between either two of the three groups (data not shown). Taken together, both MMF-AFN administration and virus treatment of the animals at the neonatal stage did not affect behaviors in their adulthood.For the analysis of the SRM we employed the Y-Maze task (Bannerman et al., In this study putative behavioral side effects of MMF-AFN anesthesia, combined with concurrent rAAV injection into the developing brain of neonates were assessed in their adulthood. For this purpose, pups of four C57BL/6N litters were anesthetized at P2, and half of the anesthetized pups received bilateral rAAV brain injections. At the age of 3 months, the matured mice were subjected to standardized behavioral tests for motor coordination, anxiety-related behavior, novel object recognition, SWM and SRM formation. In our study the neonatal MMF-AFN and brief neuosurgical intervention did not result in sever basic and cognitive behavioral impairments in animals when they reach their adulthood.Females from the FVB/N strain were employed to compare the maternal care behaviors with that of C57BL/6N dams. Our video-monitoring showed that the C57BL/6N dams exhibit less maternal care towards their pups, since two out of six C57BL/6N dams were not able to gather and maintain their offspring within the first 60 h after birth . HoweverEven though the MMF anesthesia followed with AFN-induced recovery could not be applied to P0 and P1 animals, which is most likely due to due to cardioresperitory side effects (Henke et al., Unlike the previous study showing that cryoanesthesia of neonatal mice can cause slight increase of anxiety level in adulthood (Richter et al., The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Animal Ethics Committee of the Max Planck Institute for Medical Research Heidelberg.WT conducted the experiments and analyzed the data and contributed to writing of the manuscript. UZ designed and provided the anesthesia protocol. RS contributed to the design of the experiments and writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In clinical practice, there is a need for an instrument to screen older people at risk of institutionalization.To analyze the association of frailty, walking-ability and self-rated health (SRH) with institutionalization in Finnish community-dwelling older people.n = 1087), Frailty Index (FI) (n = 1061) and PRISMA-7 (n = 1055). Walking ability was assessed as self-reported ability to walk 400 m (n = 1101). SRH was assessed by a question of general SRH (n = 1105). Cox regression model was used to analyze the association of the explanatory variables with institutionalization.In this prospective study with 10- and 18-year follow-ups, frailty was assessed using FRAIL Scale (FS) (The mean age of the participants was 73.0 (range 64.0‒97.0) years. Prevalence of institutionalization was 40.8%. In unadjusted models, frailty was associated with a higher risk of institutionalization by FS in 10-year follow-up, and FI in both follow-ups. Associations by FI persisted after age- and gender-adjustments in both follow-ups. By PRISMA-7, frailty predicted a higher risk of institutionalization in both follow-ups. In unadjusted models, inability to walk 400 m predicted a higher risk of institutionalization in both follow-ups and after adjustments in 10-year follow-up. Poor SRH predicted a higher risk of institutionalization in unadjusted models in both follow-ups and after adjustments in 10-year follow-up.Simple self-reported items of walking ability and SRH seemed to be comparable with frailty indexes in predicting institutionalization among community-dwelling older people in 10-year follow-up.In clinical practice, self-reported walking ability and SRH could be used to screen those at risk.The online version of this article (10.1007/s40520-020-01551-x) contains supplementary material, which is available to authorized users. Frailty in older adults has been described as a phenotype  as well Frailty is a dynamic state, and the possibility of slowing or reversing the cascade of decline in functional capacity with targeted multicomponent interventions has been proposed . The totSlowness is a part of the classic phenotype definition of frailty, the Fried phenotype , and oftSelf-rated health (SRH) has been shown to predict mortality , 27, insWe have earlier demonstrated the predictive ability of three frailty tools  in relation to mortality in Finnish community-dwelling older adults [. All persons born in or prior to the year 1933 (n = 1596) were invited to participate in the baseline examination that took place between March 1998 and September 1999. Of those eligible, 63 died before they were examined and 273 refused or did not respond leaving 1260 (82%) participants, 533 men and 727 women. An outlier, institutionalized before baseline at the age of 17, was excluded from the analyses leaving 1259 participants.This study is a part of the longitudinal epidemiological study carried out in the municipality of Lieto in southwest Finland . All perAt baseline the study protocol consisted of an extensive interview on demographic and socioeconomic factors and health behavior, numerous laboratory tests, and a clinical examination including a comprehensive survey of the participants’ medical records.n = 86) were excluded from the present analyses predicting institutionalization, as it was not possible to ascertain whether they continued living at home or were institutionalized in another municipality. Also participants already living in institutional care (n = 67) at baseline were excluded from the institutionalization analyses. This left us with 1106 participants.The follow-ups time were 10 and 18 years. Participants no longer living in Lieto at the end of 2016  , 16, FraThe FS is a frailty screening tool based on the Fried frailty phenotype . The oriThe FI is calculated as the proportion of individual’s deficits in relation to the total amount of deficits chosen , 3. In tPRISMA-7 includes seven self-reported items , and in Walking ability was assessed by self-reported ability to walk 400 m independently (yes/no).The information about the participants’ SRH was gathered at baseline by the question: “How would you describe your current state of health?” The answers were categorized into three groups: ‘good’, ‘intermediate’ and ‘poor’.The study was conducted according to the guidelines of the Declaration of Helsinki. The Ethics Committee of the Hospital District of Southwest Finland approved the study protocol. Participants provided written informed consent for the study.t test.At baseline, differences between men and women were tested using the Chi squared test, Fisher’s exact test or two-sample Hazard ratios (HRs) and their 95% confidence intervals for institutionalization were calculated using Cox proportional hazard models. Proportional hazards assumption was tested using Martingale residuals. The follow-up periods were calculated from the baseline measurements to the end of the follow period of 10 and 18 years or to the death of the individual. We used death as a competitive factor in the analyses.P values less than 0.05 were considered statistically significant. All statistical analyzes were performed using SAS System for Windows, version 9.4 .Firstly, unadjusted Cox regression analyzes were conducted for frailty tools, self-reported walking ability and SRH. Secondly, Cox regression analyzes for FI, FS, self-reported walking ability and SRH were adjusted for age and gender, which are items of PRISMA-7. The interaction between gender and all explanatory variables  were included in Cox regression model. The mean age of the participants was 73.0  years and 57% were women. More detailed baseline characteristics of 1106 study participants are shown in Table Three percent of participants were characterized as frail according to FS, 25% according to FI and 18% according to PRISMA-7. Figure Only 8% (9% of women and 7% of men) of the participants were self-reportedly unable to walk 400 m.n = 159) of participants rated their health as poor. The proportions of men and women in different groups of SRH  were similar: 40, 46 and 15% for men, and 41, 45 and 14% for women, respectively.Altogether 14%  women and 130 (26.5%) men were alive at the end of 2016.Of the participants self-reportedly able to walk 400 m at baseline, 65% were still living at home, 12% institutionalized and 23% deceased after 10 years (at the end of 2008). After 18 years (at the end of 2016) the proportions were 28, 28 and 44%, respectively. Of the participants self-reportedly unable to walk 400 m, 10% were living at home, 43% institutionalized and 47% deceased after 10 years. After 18 years the proportions were 0, 47 and 53%, respectively.Of the participants with good SRH at baseline, 72% were still living at home, 12% institutionalized and 16% deceased after 10 years. After 18 years, the proportions were 32, 29 and 39%, respectively. Of the participants with poor SRH, 29% were living at home, 26% institutionalized and 45% deceased after 10 years. After 18 years, the proportions were 6, 40 and 53%, respectively.When analyzing the proportion of participants institutionalized in the municipality of Lieto, we included participants who had been institutionalized before baseline. We only took into account the participants deceased by the end of 2016, to count the proportion of participants institutionalized during their lifetime. This left us with 831 participants, of which 339 were institutionalized (40.8%). There was a higher prevalence of institutionalization in women (48.9%) than in men (30.2%).The mean age of the participants at the time of institutionalization and the mean time spent living in an institution are shown in Table During the 10-year follow-up, both being frail and pre-frail according to FS and FI were associated with a higher risk of institutionalization in unadjusted Cox regression models Table . After aDuring the 18-year follow-up, being pre-frail according to FS and both being pre-frail and frail according to FI were associated with a higher risk of institutionalization in unadjusted models Table . After ap < 0.001), but not in men  during the 18-year follow-up.The association of frailty and institutionalization did not significantly differ between men and women either in 10- or 18-year follow-up by FI or PRISMA-7; using FS, being pre-frail predicted a significantly higher risk of institutionalization in women  but not in men  during the 18-year follow-up.No significant interaction was found between gender and self-reported walking ability in predicting the risk of institutionalization during either follow-ups. Poor SRH, instead, predicted a higher risk of institutionalization in women  to 25% (according to FI). Being frail according to FI and PRISMA-7 was associated with a higher risk of institutionalization in both follow-ups. Also being pre-frail according to FS was associated with a higher risk of institutionalization in 10-year follow-up. The self-reported inability to walk 400 m and poor SRH were associated with a higher risk of institutionalization in 10-year follow-up.The current estimated prevalence of frailty in European adults aged 65 years and older is 10‒20% , but theBeing frail according to FI was associated with a higher risk of institutionalization during both 10 and 18 year of follow-up also in adjusted analyses. Because of low number of subjects categorized as frail, only being pre-frail according to FS was related to higher risk of institutionalization during the 10-year follow-up. Frailty according to PRISMA-7 predicted a higher risk of institutionalization in both follow-ups. These results are in consistence with previous research , 8. In oIn our study, the self-reported inability to walk 400 m at baseline was associated with a higher risk of institutionalization in 10-year follow-up. This is similar to previous research regarding walking ability . In factWe also found that the mean age at the time of institutionalization has risen during the follow-up-period and the mean time spent living in an institution decreased. Women were institutionalized at a higher age than men and lived in an institution longer than men, reflecting that women seem to tolerate frailty and functional disabilities better than men , 6. BesiThe strengths of our study are the large sample size and a long follow-up period enabling broad generalizability to the community-dwelling older population. We also used death as a competitive factor in our analyses. The frailty tools used were validated and commonly used, although the modified versions of FS and PRISMA-7 could have had an impact on our results. Especially, prevalence of frailty was low according to FS, and it’s possible that the modifications used in our study underestimated the prevalence, and thus the association with institutionalization. The most obvious limitation to our study was that all studied explanatory variables were only assessed at baseline.To conclude, a simple self-reported item, such as self-reported ability to walk 400 m and/or self-rated health could be used in the clinical setting instead of more time-consuming frailty tools, to screen for future risk of institutionalization, and identify older persons in need of a comprehensive evaluation and possible interventions.Supplementary file1 (DOCX 23 kb)Supplementary file2 (DOCX 26 kb)Supplementary file3 (DOCX 23 kb)Below is the link to the electronic supplementary material.


Since the first appearance of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS- CoV-2) in China on December 2019, the world has now witnessed the emergence of the SARS- CoV-2 outbreak. Therefore, due to the high transmissibility rate of virus, there is an urgent need to design and develop vaccines against SARS-CoV-2 to prevent more cases affected by the virus. In this study, a computational approach is proposed for vaccine design against the envelope (E) protein of SARS-CoV-2, which contains a conserved sequence feature. First, we sought to gain potential B-cell and T-cell epitopes for vaccine designing against SARS-CoV-2. Second, we attempted to develop a multi-epitope vaccine. Immune targeting of such epitopes could theoretically provide defense against SARS-CoV-2. Finally, we evaluated the affinity of the vaccine to major histocompatibility complex (MHC) molecules to stimulate the immune system response to this vaccine. We also identified a collection of B-cell and T-cell epitopes derived from E proteins that correspond identically to SARS-CoV-2 E proteins. The in-silico design of our potential vaccine against E protein of SARS-CoV-2 demonstrated a high affinity to MHC molecules, and it can be a candidate to make a protection against this pandemic event. Symptoms such as the flu-like illness, acute respiratory distress syndrome, and clinical or radiological evidence of pneumonia in individuals needing hospitalization are considered as COVID-191. Patients diagnosed with COVID-19 are reported to have high levels of interleukin 1 beta (IL1B), interferon gamma (IFNγ), interferon-inducible protein 10 (IP10), and monocyte chemoattractant protein 1 (MCP1), likely leading to activated T-helper-1 (TH1) cell responses. In comparison, patients needing ICU admission had higher concentrations of granulocyte-colony stimulating factor (GCSF), IP10, MCP1, MIP1A, and tumor necrosis factor α (TNFα) than those not needing, suggesting a possible correlation of cytokine storm and disease intensity. Nonetheless, SARS-CoV-2 infection also resulted in the enhanced production of T-helper-2 (TH2) cytokines, such as interleukin 4 (IL4) and interleukin 10 (IL10) that inhibit inflammation that varies from that of SARS-CoV infection2. The persistent rise in patients and the high contagious rate of SARS-CoV-2 infection illustrate the immediate need to develop a safe and effective vaccine.The recent outbreak of the new virus in Wuhan City, China, originated possibly from the seafood industry has contributed to the discovery of a new strain named coronavirus and labeled the SARS-CoV-2 of the Coronaviridae family. This virus has caused severe damage and anxiety, leading to the loss of myriad individuals, impacting more than 1,000,000 people (3. Epitope-based vaccines (EVs) utilize immunogenic peptides (epitopes) to cause an immune response. The performance of the EV is calculated by the number of epitopes to be used as the foundation. Nevertheless, the experimental identification of the nominee epitopes is costly in terms of time and money. Besides, different immunological requirements need to be considered for the final choice of epitopes4.Vaccines were mostly made up of whole pathogens, either destroyed or attenuated. It may be beneficial to use peptide vaccines that are capable of generating an immune response against a specific pathogen5. Like all coronaviruses, SARS-CoV-2 comprises four viral proteins, namely spike (S) protein, a form of glycoprotein, membrane (M) protein, covering the membrane, envelope (E) protein, a strongly hydrophobic protein that covers the entire coronavirus structure, and nucleocapsid (N) protein, a structural protein that suppresses the RNA interference (RNAi) in order to overcome the host defense7  protein is a small intrinsic membrane protein actively engaged in several stages of the life cycle of the virus, such as assembling, propagation, enveloping, and pathogenesis10. This protein also slows the transport of proteins through the secretive pathway by adjusting the concentrations of Ca2+ and H+ in the Golgi and endoplasmic reticulum (ER) compartments and has been suggested to be a mechanism for immune avoidance11. The envelope (E) protein sequence was collected from a protein database and analyzed with various bioinformatics tools to identify protective epitopes. The toxicity of whole protein was analyzed, and nine epitopes including TALRLCAYCC, ALRLCAYCCN, LRLCAYCCNI, RLCAYCCNIV, LCAYCCNIVN, CAYCCNIVNV, AYCCNIVNVS, YCCNIVNVSL, and CCNIVNVSLV were identified as toxic epitopes. The predicted B-cell and T-cell epitopes were checked in terms of not coinciding with these regions. The toxic epitopes of envelope (E) protein were less than the spike protein. This finding was verified in the literature in which envelope (E) protein was explored in Severe Acute Respiratory Syndrome (SARS) in 2003 and, more recently, in the Middle East Respiratory Syndrome (MERS), the retention of this protein against the seven strains was investigated and confirmed using BioEdit Package tool17. Several experiments have explored the ability of CoVs with mutated envelope (E) protein or without mutated envelope (E) protein, concentrating mainly on SARS and MERS-CoV, as live attenuated vaccine candidates with some impressive results21. Therefore, we decided to design an effective vaccine against this protein.The electron microscope determines the properties of CoVs. They are enveloped viruses with single-stranded positive-sense RNA. The coronavirus genome size varies from 26–32 kbdefense7 . Such acproteins  are highThe amino acid sequence of envelope (E) protein of SARS-CoV-2 was collected from the NCBI virus with an accession number of QHD43418 released on 2020-01-13. The FASTA sequence was used to construct a multi-epitope vaccine against SARS-CoV-2.The sequence of “cholera toxin B (CTB) subunit [Vibrio mimicus]” was collected from the NCBI database and was used as an adjuvant.NVSLVKPSFYVYSRVK – YVYSRVKNLNSSRVPD) were chosen as protective epitopes  and two epitopes  were not carried out on “non-antigen” epitopes .Allergenicity of both B-cell and T-cell epitopes was estimated by AllerTOP v. 2.0 .Toxicity of both B-cell and T-cell epitopes was anticipated by ToxinPred, with a peptide fragment length of 10 .AJP16764.1 was selected as an adjuvant and attached to the amino terminals of the multi- epitope vaccine through PAPAP linkers as rigid linkers to improve antigen-specific immune responses22  in vivo. An instability index II was predicted to be 41.34, classifying the protein as unstable (II >40 indicates instability), but the amount of this index is really near the border. The estimated Grand Hydropathic Average (GRAVY) was −0.120. The negative attribute indicates that the protein is hydrophilic and can react with water molecules22. Furthermore, based on the PepCalc server, the solubility was predicted to be “good” in water. Based on SOLpro from ANTIGENpro, Peptide chimera was expected to be SOLUBLE with a possibility of 0.684306.Based on the Protparam database, the final peptide chimera comprised 338 amino acids  with a mBy using the Iupred2a server, the disorder regions of the final peptide, which make it unstable, were identified. The disorders are regarded in the adjuvant areas .The secondary structure of the final protein  was analThe final protein tertiary structure was built by I-TASSER server. I-TASSER provided the top 10 proteins from PDB library that had the closest structural similarity to the predicted vaccine model. The average TM-score of these ten proteins was calculated to be 0.41. We selected the best predicted model according to C-score; a confidence score to estimating the quality of predicted models by I-TASSER. It had the highest C-score of −3.96, and the RMSD was estimated to be 16.4±3.0Å.The GalaxyWEB server was used to refine the predicted model. After refinement, an evident improvement was observed in the percentage of residues in Ramachandran favored regions relative to the initial predicted model.The plot B in 23.The diagram of the predicted local similarity to target and the Z-score value of the homology model is shown in Based on the ProSA-web, the Z-score of peptide chimera was predicted to be −4.3 . This vaBased on the Procheck server, The Ramachandran plot analysis of the modeled protein reported that 72.0% of residues in the protein are in the favored regions. 22.2% of the residues were predicted to be in additional allowed regions and 2.3% were in generously allowed regions, with 9 residues (3.5%) in the disallowed regions .25.The most populated clusters were found in SFVSEETGT and HLA-A*24:02, TLAILTALR and HLA-A*24:02, VTLAILTAL and HLA-A*24:02, and TLAILTALR and HLA-A*01:01 with 997, 997, 990, and 769 cluster members, respectively.Crystal structure of HLA-A*01:01, HLA-A*24:02, HLA-A*11:01, HLA-A*03:01, HLA- B*07:02, HLA-DRADBR1*01:01 and HLA-DPB1*01:02 were retrieved from the PDB RCSB database . The PDB les were edited and cleaned from heteroatoms. PEP-FOLD2.0 from the RPBS Web Portal server was used to predict the tertiary structure of 6 epitopes of vaccine construction individually. A molecular docking study was carried out on epitopes and the whole vaccine construction with MHC alleles by the ClusPro 2.0 online server. PyMOL software was used to perform a detailed analysis of the interface of protein-protein interaction (PPI) . The weiThe docking study on the whole vaccine construction with the MHC molecules depicted one of the most probable position and orientation of the peptide within the MHC binding grooves . The actWe predicted the IL4, IL10 and INFγ inducing peptides from the 6 epitopes in the final vaccine construction via IL4pred server, IL10pred server and INFepitope server, respectively. The result predicted the SFVSEETGT epitope as an IL4 inducer and the NVSLVKPSFYVYSRVK as an IL4, IL10, and INFγ inducer and the SFYVYSRVKNLNSSRVPD as an IL4 and IL10 inducer peptide.The primary and secondary immune responses were stimulated by the C-IMMSIM server. This server simulated the immune response to vaccine candidate (without adjuvants) for three times of injection in the time steps of 1, 84, and 100. Each time step is equal to 8 hours. To make a relative comparison, we made a shuffled sequence of the vaccine candidate as a control protein, and we analyzed the results of the immune response simulation to injection of it. This shuffled sequence was employed to evaluate the significance of the vaccine sequence results, because in immune response simulation by this server, the sequence composition  is an important consideration. Finally, we found out that the results of the vaccine injection varied from those of the controls. The results of the immune response simulations are given in https://who.sprinklr.com/) as of April 13, 2020, the number of COVID-19 confirmed cases is estimated at 1,776,867 people and 111,828 deaths. Therefore, there is an immediate need to develop vaccines against this transmissible disease. There is currently no vaccination or licensed medication for humans against SARS-CoV-2. Nonetheless, further clinical trials are still needed to confirm their efficacy and safety26.Based on the World Health Organization (WHO) website , T-cells and B-cells and trigger successful responses to specific viruses12.Epitope-based vaccines offer a new strategy for the prophylactic and therapeutic use of pathogen-specific immunity30. First of all, we obtained the FASTA sequence of E protein of SARS-CoV-2 from the NCBI database. B-cell and T-cell epitopes of this protein were predicted by different servers. The epitopes were screened based on three filters of antigenicity, allergenicity, and toxicity. Therefore, we selected the protective epitopes. We merged the overlaps of B-cell and T-cell epitopes and fused them by appropriate flexible linkers. Previous studies reported that KK linkers preserve independent immune responses when they are inserted between epitopes31. Then, we linked CTB adjuvants at the terminus of epitopes by PAPAP linkers as rigid linkers to enhance the biological activities32  protein is conserved in all CoVs and covers the entire surface of SARS-CoV-2 . It has vities32 . Another22. Finally, the whole peptide chimera was analyzed for antigenicity, allergenicity, and toxicity, and it was predicted as Antigen35, Non-Allergen36, and Non-Toxin37. PI was calculated to be 9.62, which shows that the final protein is alkaline. It was predicted as “soluble” upon expression in the E.coli host. The instability index II was about 1 unit over the threshold of 40, and it resulted in considering this protein as “unstable”. However, more analysis demonstrated that the residues which are responsible for such disorders to make it unstable are in adjuvant regions not in the multi-epitope area  structures offer useful insights into their molecular activity and provide a wide variety of applications in bioscience39. The I-TASSER server modeled the tertiary structure of the final protein. Based on the Ramachandran plot, 96% of the residues of refined predicted model were found in favored and allowed regions with 3.5% outliers. Successful refining might improve the applicability of template-based models by offering more reliable structures for functional analysis, molecular design or experimental structure determination40. In the context of structural vaccinology, a molecular docking study was needed to forecast the binding affinity of epitopes to the crystallized fragment (FC) of antibodies or MHC molecules42. To analyze the affinity of the final multi-epitope vaccine to MHC molecules, we did 26 docking studies. They were carried out on the 6 epitopes of the final vaccine and the whole vaccine construction with MHC class I and class II receptors. The results of docking studies were notable and demonstrated the high affinity of the multi-epitope vaccine and its individual epitopes to MHC molecules. Then the interface of protein-protein interactions was reconsidered by a visualizations tool. At the next step of designing a multi-epitope vaccine, following an approach of systems vaccinology is beneficial in assessing the human complex immune response at different stages of biological structures43. Finally, we utilized an immune simulator server to predict the primary and secondary response of the immune system to three times of injection of the candidate vaccine. From cytokines simulation plot, we noted an increase in amounts of IL-4 and INFγ, similar to that Huang et al.2 observed in clinical features of COVID-19 patients  protein of SARS-CoV-2 to construct a chimeric peptide candidate against this pandemic disease. The results of the present study demonstrated a high affinity of this chimeric peptide to MHC molecules of the immune system, and the outputs of immune response simulation to the injection of this novel vaccine confirmed our findings. To conclude, the multi-epitope vaccine designed against E protein of SARS-CoV-2 by utilizing immunoinformatics methods may be considered as a new, safe, and efficient approach against SARS-CoV-2.http://www.violinet.org/vaxquery/) envelope (E) protein of SARS- CoV-2 can be a target to design vaccines because there is a vaccine in research status working on this protein. The amino acid sequence of the envelope (E) protein and spike protein of SARS- CoV-2 were collected from the NCBI virus (https://www.ncbi.nlm.nih.gov/labs/virus/vssi/#/) with the accession number of QHD43418 and QHR63280, respectively.Based on vaxquery database (45 (http://crdd.osdd.net/raghava/abcpred/) has analyzed the full-length envelope (E) sequence for the prediction of linear B-cell epitopes. ABCpred uses a machine learning methodology that requires fixed-length patterns for training or research, while B-cell epitopes range from 5 to 30 residues. To overcome this issue, the server sought to create data sets of fixed-length patterns from B-cell epitopes by removing or linking residues to terminals. (http://crdd.osdd.net/raghava/abcpred/).B-cell epitopes are pieces of proteins or other molecules in which antibodies (produced by B- cells) bind. Prediction methods are both time saving and cost-effective, and reliable approaches for predicting linear B-cell epitopes thus would be the first move in leading the genome-wide quest for B-cell antigens in pathogenic organism45 . ABCpred46. The first group of T-cell epitope prediction was performed using vaxign server (http://www.violinet.org/vaxign/) (47. The next two groups helper T lymphocyte (HTL) epitopes and cytotoxic T lymphocytes (CTL) epitopes were predicted individually by the IEDB database (http://tools.iedb.org/mhcii/ and http://tools.iedb.org/mhci/) (http://tools.iedb.org/main/tcell/). The other tool that is available on the IEDB database estimates peptide attachment to MHC class II molecules. This tool uses a variety of methods to predict MHC class II epitopes, including a consensus approach combining NN-align, SMM-align, and combinatorial library methods (http://tools.iedb.org/main/tcell/).T-cell epitopes are a group of peptides that can be detected by T-cell receptors after a given antigen have been processed intracellularly and attached to at least one MHC molecule and expressed on the surface of the APC as an MHC-peptide complex. Entities that have at least one MHC molecule that can most eagerly attach to allergenic amino acid sequences from an allergen and at the same time have the correct T-cell clone that can detect this MHC-peptide complex are known to be genetically susceptible to allergic reactions to this allergen. This concept can be investigated in in-silico by employing advanced statistical methods focused on sophisticated mathematics and statisticsvaxign/) . Vaxign g/mhci/) . There ihttp://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) with a threshold of 0.4 was used to predict the antigenicity of both B-cell and T-cell epitopes  with 0.4 thresholds. Likewise, ANTIGENpro (http://scratch.proteomics.ics.uci.edu/) was utilized to predict the antigenicity of peptide chimera. ANTIGENpro is an alignment-free, sequence-based, and pathogen-independent protein antigenicity predictor. The forecasts are a two-stage model based on multiple versions of the primary sequence with five machine learning algorithms. The final SVM classifier analyses the corresponding predictions and determines whether or not the protein is probable to be antigenic or not, as well as the relevant probability. ANTIGENpro is the first indicator of all protein antigenicity trained to employ reactivity data from the protein microarray analysis of five pathogens. (http://scratch.proteomics.ics.uci.edu/explanation.html#ANTIGENpro) AllerTOP v2.0 (http://www.ddg-pharmfac.net/AllerTOP/feedback.py) was used to predict the Allergenicity of both B-cell and T-cell epitopes (49.VaxiJen v2.0 (epitopes . A thresepitopes . Proteinhttps://webs.iiitd.edu.in/raghava/toxinpred/protein.php) with a peptide fragment length of 10 was used to predict toxicity of both B- and T-cell epitopes (http://crdd.osdd.net/raghava/toxinpred/). This server also was used to predict the toxicity of whole protein chimera and no fragment was predicted as toxin.ToxinPred  stabilizing the formulation of the vaccine, since physical structure of immunostimulators makes them highly unstable in aqueous solutions; 2) co-location of the vaccine antigen and adjuvant, assuring the activation of the same cells that have faced the antigen50. PAPAP linkers as rigid linkers were used to link adjuvants to epitopes (32.Selected B-cell and T-cell epitopes  were fusepitopes  in orderhttps://web.expasy.org/protparam/) was used to calculate and predict the molecular weight, isoelectronic point value (PI), in vivo, and in vitro half-life, instability index II and grand average of hydropathicity (GRAVY). ProtParam from the ExPASy server is a reliable algorithm to compute Physicochemical properties. However, it uses a single sequence per analysis through the interface.Protparam database (https://iupred2a.elte.hu/) was used to analyzing disorders which make the final protein unstable (51.The Iupred2a server (unstable . The strhttp://scratch.proteomics.ics.uci.edu/explanation.html#SOLpro) was used to predict the solubility of peptide chimerA upon overexpression. SOLpro predicts the tendency of the protein to be soluble when overexpressed in E.coli using a two-stage SVM model based on multiple representations of the primary sequence. Each first layer classifier uses a separate set of features describing the sequence as input. The final SVM classifier sums up the resulting estimates and predicts whether the protein is soluble or not, as well as the relevant probability. (http://scratch.proteomics.ics.uci.edu/explanation.html#SOLpro).SOLpro from ANTIGENpro  is another server to predict the solubility of the final protein. It is only a very rough estimation of water solubility .PepCalc server (https://prabi.ibcp.fr/htm/site/web/services/secondaryStructurePrediction) was used to predict the secondary structure of the final sequence of peptide chimera (http://www.prabi.fr/spip.php?page=services).Prabi server ( chimera . All PRAhttp://bioinf.cs.ucl.ac.uk/psipred/) is another severs to predict secondary structure employed to achieve the details of residue’s configurations. It is a very simple system of secondary prediction based on a simple neural network evaluation of PSI-BLAST-generated pro les and is capable of generating findings that place the process at the very top of the current forecasting system crop52.PSIPRED 4.0 (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) was used to carry out the homology modeling of the final protein  was used to refine the tertiary structure predicted model. In this refinement process, the server first reconstructs side chains and performs side-chain repacking following eventual overall structure relaxation through molecular dynamic simulation57.GalaxyWEB server  (https://www.ebi.ac.uk/thornton- srv/software/PROCHECK/). The Ramachandran plot is particularly useful as a test for geometric validation since φ and ψ are not part of the target function for refining. The percentage of residues found in the most favored φ and ψ regions is strongly correlated with resolution and is now reported as standard in protein structure papers, whereas specific “outlier” residues incorrect structures are used to denote either possible errors or possibly significant strained conformations59. ProSA-web (https://prosa.services.came.sbg.ac.at/prosa.php) was used to analyze the Z-score of the 3D model predicted  ; as a prredicted . The Prohttps://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal.py#forms::PEP-FOLD) performed the prediction of the tertiary structure of vaccine construct epitopes. PEP-FOLD is an online tool that is designed to model 3D peptide conformation structures in aqueous solutions for 9–25 amino acid length peptides (de novo modeling). PEP-FOLD conducts a series of 50 simulations, beginning with an amino acid sequence and returns the most critical energy and population-related conformations to be found62.PEP-FOLD 2.0 from the RPBS Web Portal server  on a grid. Then, it chose the scores of top 1000 lowest energy docked structures 70,000 rotations. These 1000 lowest energy docked structures would be processed subsequently. This set might have the potential to consist of at least some models which are close to the native structure of the complex. Then, the server clustered the 1000 rotations by finding the structure with the most “neighbors” within 9 Å IRMSD radios as the distance measure. Then, it considered this ligand and its neighbors as the “cluster center” and the “members” of the cluster, respectively. This process was repeated under remainder of the ligands to find the next clusters. Finally, the server scores the models and reported the top score ones based on the cluster size (10 most populated clusters)63.PyMOL software was used to analyze docking results. PyMOL is mostly utilized for molecular visualization by crystallographic, molecular dynamic simulation, and protein modeling software packageshttps://webs.iiitd.edu.in/raghava/il4pred/design.php), IL-10Pred server (https://webs.iiitd.edu.in/raghava/il10pred/predict3.php) and INFepitope server (https://webs.iiitd.edu.in/raghava/ifnepitope/predict.php), respectively.IL4, IL10 and INFγ inducing peptides from the 6 epitopes in the final vaccine construction were predicted via IL4pred server (http://150.146.2.1/C-IMMSIM/index.php) , respectively. We randomly shuffled the vaccine protein sequence (without adjuvants) by using the Stothard P (2000), the Sequence Manipulation Suite server (https://www.bioinformatics.org/sms2/shue_protein.html)65 to create a control group.Immune response to vaccine injection was simulated by the C-IMMSIM server (dex.php) . C-ImmSi66. The total simulation is focused on three events: 1) B-cell epitopes binding, 2) HLA class I and II epitopes binding, and 3) TCR binding, which HLA-peptide complex interaction should be presented. Such processes are independently conducted by cells described by agents and occupy a specified simulated biological amount66.The immune system simulation server mentioned here provides an opportunity to study the overall immunogenicity of the generic protein sequence in the context of its amino acid sequence


Candida albicans is the most common cause of fungal infection. The emergence of drug resistance leads to the need for novel antifungal agents. We aimed to design naphthofuranquinone analogs to treat drug-resistant C. albicans for topical application on cutaneous candidiasis. The time-killing response, agar diffusion, and live/dead assay of the antifungal activity were estimated against 5-fluorocytosine (5-FC)- or fluconazole-resistant strains. A total of 14 naphthofuranquinones were compared for their antifungal potency. The lead compounds with hydroxyimino (TCH-1140) or O-acetyl oxime (TCH-1142) moieties were the most active agents identified, showing a minimum inhibitory concentration (MIC) of 1.5 and 1.2 μM, respectively. Both compounds were superior to 5-FC and fluconazole for killing planktonic fungi. Naphthofuranquinones efficiently diminished the microbes inside and outside the biofilm. TCH-1140 and TCH-1142 were delivered into C. albicans-infected keratinocytes to eradicate intracellular fungi. The compounds did not reduce the C. albicans burden inside the macrophages, but the naphthofuranquinones promoted the transition of fungi from the virulent hypha form to the yeast form. In the in vivo skin mycosis mouse model, topically applied 5-FC and TCH-1140 reduced the C. albicans load from 1.5 × 106 to 5.4 × 105 and 1.4 × 105 CFU, respectively. The infected abscess diameter was significantly decreased by TCH-1140 (3-4 mm) as compared to the control (8 mm). The disintegrated skin-barrier function induced by the fungi was recovered to the baseline by the compound. The data support the potential of TCH-1140 as a topical agent for treating drug-resistant C. albicans infection without causing skin irritation. Candida albicans is the most frequent cause of fungal infection. It is estimated as the third most common infection in United States hospitals   while ATCC10231 is resistant to most of the antifungals, including fluconazole. ATCC90029 also can be regarded as the susceptible strain since it is only resistant to 5-FC but sensitive to most of the antifungal drugs. The emergence of resistance to antifungal drugs is usually due to the formation of biofilm, host cell residence, and filamentation, which increase the virulence of C. albicans  . There aalbicans . The abiC. albicans. To explore the fungicidal mechanism of the compounds, morphological observation, total DNA, RNA, protein, and hypha-related genes were assessed to determine the mode of action on C. albicans. Finally, we compared the antifungal activity between the compound and 5-FC in the in vivo skin mycosis mouse model. Here, we demonstrated that topical delivery of the angular naphthofuranquinone with the hydroxyimino group (TCH-1140) remarkably mitigated abscess and the associated C. albicans burden.The intracellular fungi killing was examined by employing keratinocytes and macrophages as the host cells infected by 1H NMR, 13C NMR, and electrospray ionization mass spectrometry as exhibited previously.We synthesized 14 naphthofuranquinone-related compounds as shown inC. albicans employed in this work were ATCC90029 and ATCC10231 obtained from the Food Industry Research and Development Institute . Both species were drug-resistant isolates.The strains of C. albicans was 16 h. MFC was detected as the lowest compound concentration for killing ≥99.9% of the fungi. The detailed procedures were represented in the previous study  and incubated for 48 h at 37°C. The absorbance of each well was detected at 600 nm to determine the growth of C. albicans in a real-time mode.The eradication of C. albicans (OD600 = 0.7) on the agar plate. Naphthofuranquinones at 0.7-2.9 μM (10 μl) were loaded onto the plate. After incubating for 12 h at 37°C, the clear zone diameter without fungi was calculated.This assay was performed by plating C. albicans by treatment of naphthofuranquinones were monitored by Live/Dead BacLight® kit. C. albicans was grown to OD600 = 0.1, and then treated with the compounds at 23.5-93.8 μM for 4 h. The strains stained by SYTO9 and propidium iodide (PI) were analyzed by fluorescence microscopy and flow cytometry. The detailed procedures were described earlier (The viability and death of  earlier .® dish by incubating the microbes (OD600 = 0.1) in 1% glucose at 37°C for 24 h. The compounds at 11.6 μM were then incorporated into the biofilm for 24 h. The recovered C. albicans inside and outside the biofilm was loaded in an agar plate for 24 h to calculate CFU.The biofilm was established in a Cellview4 and 1 × 105 cells/well, respectively. The MTT assay was reported previously (The vulture method for keratinocytes (HaCaT) was described in detail in our previous study . THP-1 ceviously . The expC. albicans in DMEM at 37°C and 5% CO2. The naphthofuranquinones dosed at 93.8 and 22.5 μM was used to treat HaCaT and C. albicans, respectively. After incubation, the cells were detached, washed with cold PBS, and treated with Annexin V-FITC and PI as suggested by the manufacturer (R&D Systems). The cells incubated with DMEM were employed as the control.Analysis of Annexin V-fluorescein isothiocyanate (FITC) and PI cell staining was performed after 24 h of incubation of HaCaT or C. albicans antibody/Alexa Fluor® 488 goat anti-mouse IgG was utilized to stain the host cell nucleus and the fungi, respectively. The detailed processes were shown in the previous work (Keratinocytes and macrophages differentiated from THP-1 were used as the host cells to estimate killing by naphthofuranquinones (11.7 μM). The fungal survival inside the host cells was appraised by colony-forming unit (CFU) counting and observed by confocal microscopy. DAPI and anti-ous work .Candida albicans morphology after treatment by naphthofuranquinones was monitored by scanning electron microscopy (SEM). The microbes at OD600 = 0.1 were treated with compounds at 11.7 μM for 24 h. The detailed processes of morphological observation were described earlier , and Bio-Rad protein assay kit, respectively.efg1, ume6, and hgc1 in C. albicans after treatment of the compounds (11.7 μM). The RNA extraction and cDNA synthesis were conducted as described before  to analyze d before . The cDNin vivo experiments. All protocols were performed in strict accordance with the recommendations set forth in the Guidelines for the Institutional Animal Care and Use Committee of Chang Gung University.Eight-week-old male Balb/c mice were used in the 6 CFU ATCC10231 strain. The topically applied lead compound or 5-FC (2.9 mM) with a volume of 0.1 ml was administered onto the infection region every 24 h for 7 days. The skin-surface appearance was visualized by phenotypic and microscopic images after 7 days. The infected area was excised for homogenization. Fungus CFU in the infected site was estimated. TEWL was evaluated by Tewameter from 0 to 7 days post-injection.All mice were prepared by shaving the dorsal fur. The mice were intradermally injected with 100 μl of the 5 × 10The mouse skin specimen was immersed in 10% formaldehyde and embedded in paraffin. The specimen was cut into a 5-μm thickness for hematoxylin and eosin (H&E) staining. We also examined immunoglobulin (Ig)G, interferon (IFN)-γ, interleukin (IL)-17, and Ly6G in the skin by immunohistochemistry (IHC). The skin sections were incubated with the related antibodies for 1 h, then incubated with biotinylated donkey anti-goat IgG for 20 min. The slices were visualized by optical microscopy.∗), skin-surface pH, and H&E-stained histology.The vehicle with naphthofuranquinones or 5-FC (2.9 mM) was topically applied on the back of the mice for 5 days. The vehicle was replaced by a new one each day. The skin was examined for its microscopic appearance, transepidermal water loss (TEWL), erythema . The significant difference between the various groups was checked by the Kruskal–Wallis test. The We designed naphthofuranquinones conjugated with imine moiety with angular or linear structures to test the antifungal effect. As a first step to screen the activity, we sought to identify the MFC against ATCC90029 . Four anC. albicans. The time-killing study offers information regarding the extent and rate of antifungal activity . On the other hand, 5-FC at 23.5 μM significantly decreased the fluconazole-resistant strain (ATCC10231), whereas fluconazole could not. A further decrease of the live ATCC10231 burden  was observed for naphthofuranquinones than 5-FC. The live/dead images of C. albicans treated with higher doses (46.9 and 93.8 μM) showed some SYTO9 reduction by the positive controls  and THP-1 (macrophages) was assessed using MTT and cell counting kit-8 (CCK-8) assays. The control in this experiment was the cells treated with DMSO vehicle . The positive control drugs and TCH-1140 did not exhibit cytotoxicity against both cells (viability > 80%) in the MTT assay  area is increased by this compound  and DAPI (blue) for monitoring by confocal microscopy. The images indicated that both fungal strains in HaCaT or THP-1 showed significant hyphae. Some fungi were present outside the host cells, suggesting a possible escape from the cells during a 4-h incubation. These filamentous hyphae in keratinocytes were unaffected by 5-FC and fluconazole , or skin surface pH was changed after administration of the compounds . The in vivo effect of the lead compound in the cutaneous candidiasis mouse model together with acceptable safety reinforce the potential as a candidate in the development of an antifungal agent.O-acetyl oxime (TCH-1142) moieties at the 4-position showed the greatest potency. All others had negligible activity. The linear naphthofuranquinone structures totally lost the antifungal activity. The conjugation of the hydrazino group at 4-position also eliminated the activity. We selected TCH-1140 and TCH-1142 for further comparison with 5-FC and fluconazole. The MIC of TCH-1140 and TCH-1142 against ATCC90029 was 1.5 μM (0.32 μg/ml) and 1.2 μM (0.31 μg/ml), respectively. These values were greatly lower than the other naphthoquinone derivatives reported earlier such as 2-methyl-5-hydroxynaphthofuran-4,9-dione (2-8 μg/ml), phenanthrenequinone (60 μg/ml), 2-[iodomethyl]-2,3-dihydronaphthofuran-4,9-dione (12 μg/ml), and plumbagin (8 μg/ml)  and 8 μg/ml) , althougkin rash . Flucona failure . It is a≥ 210 μM . We reco≥ 210 μM . Our res≥ 210 μM . Our timC. albicans by naphthofuranquinones compared to that by fluconazole could be due to the inhibition of the efflux pump regulated by some genes such as mdr1. The mechanisms of C. albicans death elicited by the lead compounds are still not completely understood. It should also be noticeable that the C. albicans growth was elevated after a 30-h treatment of lead compounds at 2.9 μM in the time-response test. The strains might gain resistance toward the compounds after a long-term intervention. Further study is needed to explore the details.The naphthofuranquinones evoked fungal wall and/or membrane disturbance based on SEM, indicating the occurrence of compromised membrane integrity. However, this damage could be regarded as mild since PI could not permeate into the cytoplasm as observed in the live/dead assay. Naphthofuranquinone intervention interfered with the total DNA, RNA, and protein synthesis in the fungi. The lead compounds might penetrate the membrane into the fungal cytoplasm, producing killing by DNA replication inhibition but not the direct membrane disintegration. Drug efflux is an important resistance mechanism, especially for fluconazole resistance . The supCandida species are difficult to treat as they invade host cells. This is a manifestation of drug resistance. More than 60% of the antimicrobials failed to manage intracellular microbes (C. albicans, most of the fungi survive inside the macrophages with only a portion of the population being killed (C. albicans without affecting host-cell viability. This intracellular elimination by naphthofuranquinones was better than 5-FC and fluconazole. The lead compounds did not arrest C. albicans growth in the macrophages. Rather, they drove the fungal transition from the pathologic hypha to the pseudohypha or yeast. The naphthofuranquinones can be anti-virulence agents. Engulfment of yeast C. albicans by keratinocytes and macrophages induces the production of hypha, which exerts a virulent action (C. albicans from the cells (microbes . Both kemicrobes . Althougg killed . Here, wt action . The hyphe cells . This waefg1, ume6, and hgc1 are transcription regulators associated with hypha formation. The transcription factor efg1 functions to promote filamentous growth (ume6 is a downstream target of hypha-growth signaling (efg1 and upstream of hgc1. The experimental data demonstrated that TCH-1140 suppressed the transcription of these genes. The upregulation of efg1 by 5-FC was surprising. efg1 appears to be an upstream regulator in the network of filamentation. Besides the involvement of the hypha form, efg1 also acts as the regulator of cell wall proteins, adhesion, aspartic protease, and antifungal resistance (efg1 upregulation in response to the environmental change because of the complex role of this gene in adapting morphogenetic transition.The development of resistance to antifungal drugs often occurs in filamentous fungi. s growth . ume6 isignaling . This kesistance . 5-FC stC. albicans. Hyphal elements are the main structures embedded in mature C. albicans biofilm (C. albicans inside the mature biofilm. Although a statistically significant CFU reduction was obtained, this killing by the compounds was still limited. The high cell density inside the biofilm has markedly lowered susceptibility to all drugs (C. albicans from biofilm displays increased virulence, filamentation, and drug resistance, which are responsible for invasive infection (C. albicans inside the biofilm but also against the microbes dispersed from the biofilm. The dispersal inhibition by TCH-1140 and TCH-1142 was superior to that in the positive controls.Both morphogenesis and biofilm are the important virulences of drug-resistant  biofilm . The bio biofilm . 5-FC an biofilm . The ant biofilm . We demoll drugs . The disnfection . The napC. albicans strains resistant to current agents has contributed to an increase in the skin infection reported in humans. Our results manifested that TCH-1140 was superior to 5-FC for ameliorating drug-resistant C. albicans-induced infection and inflammation. TCH-1140 also recovered skin-barrier capacity disrupted by fungi. C. albicans infection is a result of a coordinated battle with host cells. The recognition of C. albicans by skin cells and innate immune cells induces the production of proinflammatory mediators. A previous study (C. albicans infection induced candidiasis with IL-17 and IFN-γ expression. Th17 cells are vital in the defense against fungal infection (C. albicans through neutrophil recruitment for killing the pathogens by releasing antimicrobial peptides or phagocytosis (The emergence of us study  proved tnfection . IL-17 oocytosis . The phaocytosis . We alsoC. albicans (C. albicans results in the decrease of IFN-γ, the Th1-related response (IFN-γ is chiefly secreted by keratinocytes to play a key role in the defense against albicans . TCH-114response . Gow et C. albicans. Both compounds were active against yeast, filamentous, and biofilm C. albicans strains that were resistant to 5-FC or fluconazole. The lead compounds could deliver into the host cells, leading to a marked clearance of intracellular fungi. The resistant fungus infection and the associated inflammation in the skin were mitigated by TCH-1140. Our findings suggested that TCH-1140 could be a candidate for the treatment of skin mycosis. There is room for further structural modification in naphthofuranquinones for lead optimization of the potentiating activity. Naphthofuranquinone analogs do warrant further investigation as a topical agent for treating fungus-infected skin lesions.After screening a series of naphthofuranquinones, TCH-1140 and TCH-1142 were proven to be the most potent for eradicating drug-resistant The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of Chang Gung University.J-YF initiated the study and drafted the manuscript. K-WT involved in the design of all experiments. S-HY, S-CY, and Y-CY carried out the experiments. AA analyzed data and wrote the manuscript. S-CY supervised the entire project. C-HT reviewed critically and approved the final manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There is increasing interest in the utility of gamma-band activity for assessing various brain functions, including perception, language, memory, and cognition. The auditory steady-state response (ASSR) involves neural activity in the brain elicited by trains of a click sound, and its maximum response is obtained at 40 Hz (40-Hz ASSR). Abnormalities of the 40-Hz ASSR are also widely reported in patients with schizophrenia. Thus, the test-retest reliability of the ASSR is important for its clinical and translational application. However, there are only limited studies reporting the short-term reliability between acquisitions at two time points made using the same electroencephalogram (EEG) system. Furthermore, the long-term reliability between multiple EEG systems and the reliability of spontaneous gamma activity are unknown but are crucial for multicenter collaborative research.We examined the long-term test–retest reliability of 40-Hz ASSR oscillatory activities indexed by the phase locking factor (PLF), evoked power, and (non-phase-locked) induced power between two clinical 19-electrode EEG systems [recorded twice for EEG-1 (time1 and time2) and EEG-2 (time3 and time4)] at four time points from 14 healthy controls over a duration of 5 months. Test-retest reliability was examined using intraclass correlation coefficients (ICCs).Both PLF and evoked power showed good to excellent ICCs (>0.60), mainly in the Fz-electrode, both within each EEG system—EEG-1  and EEG-2 —and between the two EEG systems . In contrast, induced power showed the highest (excellent) ICC between the two EEG systems (ICC = 0.95) mainly in the Cz-electrode. For PLF, the Fz-electrode showed better test-retest reliability across all EEG recordings than the Cz-electrode , whereas we found similar excellent reproducibility across all EEG recordings from both electrodes for evoked power  and induced power .The 40-Hz ASSR oscillatory activities, including induced power, showed excellent test-retest reliability, even when using different EEG systems over a duration of 5 months. These findings confirm the utility of the 40-Hz ASSR as a reliable clinical and translatable biomarker for multicenter collaborative research. Gamma-band 30−100 Hz) neural oscillations are implicated in normal perception, cognition, and memory, while recent empirical studies suggest that gamma-band activity is disrupted in patients with schizophrenia 00 Hz neu. GABAergMany studies have shown a decreased evoked power value and phase synchronization of 40-Hz ASSR in both the early and chronic phases of schizophrenia –13. In aOther mental conditions such as bipolar disorder or affective psychosis show similar ASSR deficits, indicating that ASSR may provide a useful index for distinguishing these conditions from major depressive disorder , 16. HowTo achieve such large-sample collaborative studies, it is critical to confirm the repeatability and reproducibility of the ASSR. Legget et al. confirmed the reproducibility of the 40-Hz ASSR within the same device using an electroencephalogram (EEG) and a magnetoencephalogram (MEG) in healthy controls , while RTherefore, in the present study, we examined the long-term test-retest reproducibility of 40-Hz ASSR oscillatory activities, including induced power, by performing two EEG oscillatory measurements at four time points using two EEG systems to assess the utility of the 40-Hz ASSR index.Fourteen healthy adults  participated in this study. All participants were screened for normal hearing. The inclusion criteria were: 1) no history of neurological illness including epilepsy, 2) no history of major head trauma, 3) no history of alcohol/drug dependence or abuse, 4) no history of electroconvulsive therapy, and 5) verbal IQ > 75. Participants were also screened using the Structured Clinical Interview (non-patient edition), and no subjects or their first-degree relatives were found to have an Axis-I psychiatric disorder. All subjects were recruited from the local community in Fukuoka metropolitan areas. Experimental procedures were approved by the Kyushu University Institutional Review Board for Clinical Trials  and conformed to the Declaration of Helsinki. Before the experiments, written informed consent was acquired from each subject after a detailed explanation of the study.To examine the long-term test-retest reproducibility of the 40-Hz ASSR using different EEG systems, EEG measurements at four time points were repeatedly performed with two EEG systems . The interval times between the four EEG measurements were: time1−time2 = 31.2 ± 26.9 days, time2−time3 = 73.0 ± 34.9 days, and time3−time4 = 42.7 ± 28.9 days. Hence, the EEG data were collected four times over a duration of around 5 months. The two EEG systems used the same electrode format and number but had different methods for measuring the time constant  and different anti-aliasing filter systems. All continuous EEG data were acquired using a 19-electrode silver/silver chloride (Ag–AgCl) passive electrode (Nihon-Koden Co.) with a standard 10–20 system electrode placement . Electrohttps://mne.tools/dev/index.html). An offline 1–100 Hz bandpass filter and notch filters of 60 Hz were applied to the EEG data. Ocular and cardiac artifacts were removed using independent component analysis. Artifact-free EEG data were average-referenced, and epochs of 1,100 ms were created, starting at 400 ms prior to stimulus onset and lasting for 700-ms post-stimulus onset (epochs with peak-to-peak amplitude > 200 mV were rejected). The number of accepted trials used for further analyses were: time1, 149.7 ± 0.5; time2, 149.5 ± 0.5; time3, 145.4 ± 1.2; time4, 146.1 ± 1.0. The 40-Hz ASSR was analyzed using the Morlet waveform transform (f0/σf = 7). We calculated the phase locking factor (PLF), evoked power, and induced (non-phase-locked) power that were applied to the single-trial epochs in 0.1-Hz steps from 4−100 Hz at each time point from −400 ms (prior to stimulus onset) to 700 ms (post-stimulus onset). PLF measures the variance of phase across single trials and ranges from 0 (random distribution) to 1 (perfect phase locked). Evoked power measures the power of the average evoked potential in which the contribution of non-stimulus locked activity is minimized. Induced power was measured by subtracting the evoked power from the averaged power of the single-trial potential. The mean PLF and evoked power from 100 to 500 ms were averaged across 10 Hz bands (35–45 Hz). The mean (non-phase-locked) induced power in the baseline period (−400–0 ms) was calculated by averaging the power across 70-Hz bands (30–100 Hz).EEG data were preprocessed using MNE Python  showed good to excellent ICCs (> 0.60), mainly in the Fz-electrode, within both EEG-1  and EEG-2 . Additionally, the Fz-electrode showed higher ICCs than the Cz-electrode for PLF  and evoked power  between the two EEG systems (EEG-1 vs. EEG-2).Induced power (non-phase-locked activity) showed excellent ICCs (> 0.75) in both electrodes within both EEG-1  and EEG-2 . Moreover, in contrast to PLF and evoked power, the Cz-electrode showed the highest ICC for induced power between the two EEG systems .Similarly, in the test to evaluate reliability across all EEG recordings, the Fz-electrode showed higher test-retest reliability than the Cz-electrode for PLF . In contrast, we found similar excellent test-retest reliability across all EEG recordings in both electrodes for evoked power  and induced power .A summary of the statistical results is shown in To the best of our knowledge, this is the first report of long-term excellent test-retest reliability at multiple time points for 40-Hz ASSR oscillatory measurements. These findings are in line with previous reports , 18, 28 r or ρ = 0.31−0.81), the N1 amplitude (r or ρ = 0.09−0.75)  –32, and 09−0.75) –37. It sImportantly, our results also demonstrated that the Fz electrode had the highest reliability or internal consistency for both PLF and evoked power when evaluating the 40-Hz ASSR using the 19-electrode standard 10–20 clinical EEG system. Our results are comparable with previous reports of excellent test-retest reliability of the 40-Hz ASSR at the Fz or FCz electrodes , 28. In r = 0.89; normalized evoked power, r = 0.90). In that study, the authors also conducted source level signal-space projection analysis within the auditory cortex and found a similar test-retest reliability . Recently, Roach et al. demonstrated fine reproducibility of the 40-Hz ASSR at the Fz electrode using the G-coefficient in both healthy subjects and schizophrenia patients  tone is widely used in audiology and was found to be more pleasant and less affected by subjects’ attention than the click trains . By contEnhancing attention to the ASSR task reportedly increased the 40-Hz ASSR oscillatory indices , 44, whiA potential caveat in our study is that both EEG systems were from the same company, and although there are differences such as the time constant and filter system, the basic amplifier structure and electrode type were the same. Thus, there is a need to test reproducibility between entirely different EEG systems, to eliminate some of the variability that could stem from using different companies’ EEG systems. Another potential limitation is that our study design could not randomly assign EEG-1 and EEG-2 during the four time points because of the replacement. Nevertheless, our results indicate that switching from one EEG system to its next generation one within the same environment (same stimulus presentation setup and other aspects of the setting) has little to no effect on the ASSR-EEG data recorded. Recently, large-sample collaborative studies are becoming crucial to characterize healthy and clinical population groups in psychiatric research, and reproducibility tests at different centers using traveling subjects would confirm our results. Importantly, if traveling subjects are not available, multicenter studies may also greatly benefit from adapting statistical models correcting for the effects of each site e.g., . It shouOur findings confirm the excellent long-term reliability and utility of 40-Hz ASSR oscillatory activities, including induced power, even when different EEG systems were used. Thus, we propose that 40-Hz ASSR oscillatory measures are one of the best neurophysiological indices for future collaborative research, which is essential for clinical and translational applications.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Kyushu University Institutional Review Board for Clinical Trials. The patients/participants provided their written informed consent to participate in this study.YH and IN had full access to all of the data in the study, and take responsibility for the integrity of the data and the accuracy of the data analysis. All authors were involved in concept and design and performed critical revision of the manuscript for important intellectual content. IN, ST, and YH performed acquisition, analysis, or interpretation of the data, drafted the manuscript, and performed statistical analysis. TO and YH performed supervision.This work was supported by JSPS KAKENHI  and in part by AMED under Grant Number JP20dm0207069 (TO). This work was also partly supported by the Fund for Pharmacopsychiatry Research from the Senshin Medical Research Foundation (YH), Fund for Takeda Science Foundation (YH), Research Grant Award (YH) from UBE Industries Foundation, and the SIRS Research Fund Award (YH).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.